Facile Photoimmobilization of Proteins onto Low-Binding PEG-Coated

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Facile Photoimmobilization of Proteins onto Low-Binding PEGCoated Polymer Surfaces Esben Kjær Unmack Larsen, Morten Bo Lindholm Mikkelsen, and Niels B. Larsen* Department of Micro- and Nanotechnology, DTU Nanotech, Technical University of Denmark, Ørsteds Plads 345E, DK-2800 Kgs. Lyngby, Denmark S Supporting Information *

ABSTRACT: Immobilization of proteins onto polymer surfaces usually requires specific reactive functional groups. Here, we show an easy one-step method to conjugate protein covalently onto almost any polymer surface, including low protein-binding poly(ethylene glycol) (PEG), without the requirement for the presence of specific functional groups. Several types of proteins, including alkaline phosphatase, bovine serum albumin, and polyclonal antibodies, were photoimmobilized onto a PEG-coated polymer surface using a water-soluble benzophenone as photosensitizer. Protein functionality after immobilization was verified for both enzymes and antibodies, and their presence on the surface was confirmed by X-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy. Conjugation of capture antibody onto the PEG coating was employed for a simplified ELISA protocol without the need for blocking uncoated surface areas, showing ng/ mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features.

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INTRODUCTION Conjugation of proteins onto polymer surfaces is important for many applications including cell selection,1,2 lab-on-a-chip systems,3 microarrays,4 targeted nanoparticles,5 and enzymelinked immunosorbent assays (ELISA).6 Binding of protein is normally performed on a surface containing chemical groups reactive to the protein chemistry, like carboxylic acids or amines. However, most polymer surfaces do not natively present such groups, which then have to be introduced before conjugation with the protein.7−10 The ability to conjugate proteins onto a native surface can be achieved with photoimmobilization where the photosensitizer molecule normally is attached to the surface or to the protein.11−17 Surfaces to be modified with a photosensitizer need chemically reactive groups to conjugate the photosensitizer, which makes the procedure complicated.18−21 Alternatively, proteins to be conjugated with photosensitizers also require chemically reactive groups to conjugate the photosensitizer onto the protein, and these groups can be involved in the protein’s function.12,22−26 Moreover, both methods require a multistep procedure to conjugate the photosensitizer onto the surface or the protein, which limits the general use of the methods. Some of the prior studies demonstrated the conjugation of a protein to a polymer surface by mixing the protein with a photosensitizer before UV exposure. Ito et al. mixed polymer, protein, and photosensitizer and dried the solution onto a surface before UV illumination. The process resulted in entrapment of the protein in a thick polymer layer.13,17 © 2014 American Chemical Society

Nahar et al. reported protein binding via a thermochemical reaction after drying of the photosensitizer solution on a surface, yielding a 2-fold increase in protein concentration on the surface compared to passively adsorbed protein.11 Both approaches require complete drying of the reagent solution before the covalent coupling can be performed. Dried reagents may form thick films that are incompatible with closed systems, for example, microfluidics, and efficient drying in such systems may in itself be challenging. Additionally, dried reagents are often deposited in a spatially inhomogeneous pattern due to a complex interplay of solvent convection and evaporation, one example being the commonly observed “coffee ring” patterns.27 Special care has to be taken when engineering protein attachment to polymer surfaces since most polymer surfaces are prone to nonspecific protein adsorption that can lead to unstable devices and low signal-to-noise ratios in analytical devices.28 Protocols for the analytically important application of ELISA assays generally alleviate this problem by passively adsorbing a nonadhesive (blocking) protein, usually albumin, after the initial passive adsorption of the capture antibody.29−31 Blocking proteins are often adequate in static analysis systems intended for immediate use, but they may be removed by flowing liquid in a dynamic system or may interfere with the assays performed, for example, in cell experiments.28 Thus, Received: November 27, 2013 Revised: February 11, 2014 Published: February 13, 2014 894

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Figure 1. Benzoyl benzylamine hydrochloride (Bz) absorbs UV light and abstracts hydrogen from carbon−hydrogen containing molecules present either on surfaces or in solution, including Bz itself. The radicals can recombine and bind the molecules onto the surface. Bz coupled to a surface via its methylene group can be activated by subsequent UV light exposure to couple dissolved reagents. the emitted photoelectrons at pass energies of 200 or 50 eV for survey or high resolution spectra, respectively. Elemental compositions were determined from acquired survey scans using the instrument manufacturer’s Avantage software package. Coating of Polymer Surfaces. PEG coating solutions consisted of 10 mM NHS-PEG and 10 mM Bz in PBS. The reactant solution was added onto the polymer substrate surface, and the surface was exposed to UV light using a custom built photoreactor with a broad emission maximum from 330 to 380 nm (Philips Cleo S-R fluorescent tubes) at an intensity of 18 mW/cm2. The exposed samples were flushed three times with water and ethanol. Alkaline Phosphatase Conjugation. A PEG coating was made as described in Coating of Polymer Surfaces in a 96-well polystyrene microtiter plate. In a second step, the enzyme was conjugated to the PEG-coated surfaces by Bz: All combinations of AP (20 and 100 μg/ mL) and Bz (0, 10, 100, and 1000 μg/mL) dissolved in PBS were added to the wells and exposed to UV light for 15 min. After washing with water three times, 100 μL of AP substrate was added to each well. The color change was measured with 2 min interval using a plate reader (Victor3, Perkin−Elmer, MA). The change in color was correlated to a standard curve made with known AP concentrations in solution to calculate a bound mass of AP, with the caveat that actual activity of the bound AP is unknown. ELISA with Conjugated Capture Antibody. The surfaces of 96well polystyrene microtiter plates were coated with PEG as described above. Between all steps the surface was washed three times with PBS + 0.05% v/v Tween 20 and then three times with PBS. Rabbit antimouse IgG served as capture antibody and was conjugated onto the PEG surface with Bz in various concentrations using 100 μL of solution per well. IgG from mouse serum served as antigen, and 100 μL of IgG in PBS at a range of concentrations was added to each well for 1 h. Goat antimouse IgG-HRP served as detection antibody, and 100 μL of 1 μg/mL IgG-HRP in PBS was added for 1 h. Thereafter, 100 μL of HRP substrate was added to each well. The color change was measured at 2 min intervals on the plate reader. The change in color was correlated to a standard curve made with known IgG-HRP concentrations in solution. For the human CCL19 (hCCL19) ELISA system, anti-hCCL19 antibody from goat was used as capture antibody, hCCL19 was used as antigen, biotinylated anti-hCCL19 antibody from goat was used as detection antibody, and HRPstreptavidin was used for detection by enzymatic conversion, according to the protocol provided by the manufacturer. Projection Lithography System. A Zeiss AxioVert 35 M inverted microscope (Carl Zeiss, Oberkochen, Germany) was converted to a projection lithography system by exchanging the field diaphragm of the Köhler illumination system with a holder for 20 mm circular chrome-on-glass shadow masks.38 The microscope was equipped with a 100 W mercury arc-discharge lamp and a 365 ± 5 nm bandpass filter. The PEG-coated polystyrene samples were exposed through a 20× objective (NA 0.5), which produced a demagnification of the mask motif by a factor of 13.0 and an irradiance of 720 mW/cm2. The power density was measured at 365 nm using an OAI 306 power meter (OAI Instruments, San Jose, CA) with the probe located at the sample plane and adjusting for the difference in spot size and photodetector probe size in calculating the actual power density. Shadow masks were

there is a dual need for minimizing nonspecific protein adsorption and maximizing specific protein coupling in a stable format by a method applicable to many types of polymer surfaces. Here, we present an easy one-step photochemical method to covalently bind proteins to a polymer surface that does not contain any specific chemically reactive groups. The surface is pretreated with poly(ethylene glycol) (PEG) together with a water-soluble benzophenone in a one-step reaction to form a base layer with low binding affinity. We have previously demonstrated the ability of this particular layer type to limit adsorption of a wide range of organic molecules, including small drugs, DNA, peptides, and proteins, including antibodies.32 This is in agreement with the vast body of prior work showing how PEG coatings significantly limit the adsorption of proteins in many applications.33−37 Targeted proteins are subsequently covalently bound to the PEG layer in an equivalent photoinitiated reaction using the water-soluble benzophenone. The method is demonstrated for different classes of proteins, including alkaline phosphatase (AP, test case for retained enzymatic activity), bovine serum albumin (BSA, a serum protein), and antibodies (IgG, test case for retained antigen recognition in an ELISA assay) in aqueous buffer. Flood UV illumination results in a homogeneous protein coating, while spatially patterned UV illumination in a projection lithography system produces micrometer-sized protein patterns on the underlying PEG base layer.

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MATERIALS AND METHODS

Materials. 4-Benzoyl benzylamine hydrochloride (Bz) was purchased from Fluorochem (Hadfield, U.K.). Fluorescein-conjugated bovine serum albumin (BSA-fluorescein), phosphate buffered saline (PBS), horseradish peroxidase (HRP), antimouse IgG (whole molecule) antibody produced in rabbit (cat. no. M7023), IgG from mouse serum (cat. no. I5381), anti-mouse IgG (whole molecule)− peroxidase antibody produced in goat (A4416), 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) liquid substrate system (HRP substrate), alkaline phosphatase, and alkaline phosphatase yellow (pNPP) liquid substrate (AP substrate) was purchased from SigmaAldrich (St. Louis, MO). OMe-PEG-NHS (750 Da, cat. no. PEG1166.0001, NHS-PEG) was purchased from IRIS Biotech (Marktredwitz, Germany). An ELISA assay (cat. no. DY361) for human cytokine CCL19 was acquired from R&D systems (Minneapolis, MN). All water used was from a Millipore Milli-Q purification system (Boston, MA). Nunc 96-well polystyrene (cat. no. 260860) and 96-well optical bottom plates (black polystyrene side, cell culture treated foil at bottom, cat. no. 165305) was purchased from Fisher Scientific (Roskilde, Denmark). X-ray Photoelectron Spectroscopy (XPS). XPS was performed on a K-Alpha spectrometer (Thermo Fisher Scientific, U.K.) using a 400 μm wide monochromatized Al Kα X-ray spot with collection of 895

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fabricated by standard photolithography and lift-off of 100 nm chromium on 500 μm borosilicate glass and cut out using a highintensity 1064 nm laser. Patterned Protein Conjugation. The 96-well black polystyrene plates were coated with PEG as described above. A total of 50 μL of Bz (100 μg/mL) and BSA-fluorescein (100 μg/mL) were added in PBS to one of the wells and exposed for 20 min in the projection lithography system using a mask motif containing our university logo in different sizes. The exposed surfaces were subsequently washed with water, and the attached protein was visualized on a confocal fluorescence microscope (LSM 5, Carl Zeiss) using illumination at 488 nm and collecting emitted fluorescence from 505 to 545 nm.

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RESULTS AND DISCUSSION Alkaline Phosphatase Enzymes Photoimmobilized on Low-Binding PEG Retain Their Activity. Many proteins adsorb passively on polymer surfaces and it is very difficult to remove these proteins without damaging all the protein in the process. Previously, we have developed a polymer coating that can minimize protein adsorption on a range of different polymer substrate surfaces, including polystyrene.32 The PEG coating was introduced on the polystyrene well surfaces of the microtiter plate by UV illumination of an aqueous solution of the photosensitizer 4-benzoyl benzylamine hydrochloride (Bz) and NHS-PEG (Figure 1). Under UV illumination, the benzophenone will form a biradical that can abstract hydrogen from any carbon−hydrogen containing molecules on a surface or in solution.39,40 These activated radicals can then recombine with other radicals on the surface or in solution to form a covalent bond. The use of a water-soluble photosensitizer avoids competing coupling reactions at the surface and in solution of activated organic solvent molecules present at much higher concentrations than the dissolved polymer. Additionally, an aqueous environment minimizes the risk of perturbing or denaturing dissolved target biomolecules during the next protein immobilization step. A solution of the enzyme alkaline phosphatase (AP) and Bz was dispensed onto PEG-coated microtiter well surfaces and illuminated by UV light (30 min) to investigate if a protein could be covalently bound while retaining some or all of its biological function (Figure 1). Figure 2 shows the measured enzymatic activity after treatment with and without UV light on a polystyrene surface as received (“On PS”) or on a PEGcoated polystyrene surface (“On PEG-Coated PS”). The amount of active conjugated protein increases strongly at the highest Bz concentration upon UV illumination of PEG-coated polystyrene. Very small amounts of protein are conjugated in the absence of UV illumination on the samples with an underlying PEG layer. In contrast, some nonspecific protein adsorption is observed on the native polystyrene surfaces, with or without UV illumination. The amount of bound enzyme is calculated as the amount of dissolved enzyme having an equivalent activity. Thus, this value does not distinguish between smaller amounts of bound enzyme of full activity or larger amounts of bound enzyme of reduced activity. Photoimmobilization of IgG on Low-Binding PEG Obviates Blocking in ELISA. We used the parameter set for the successful and simple photoimmobilization of active AP as a starting point for immobilization of active antibodies for ELISA type assays in polystyrene microtiter plates. The relevant range of conjugation parameters were established by varying the Bz concentration, the capture antibody concentration and the UV illumination time (Electronic Supporting Information, Table S1). Bz concentrations smaller than 100 μg/mL allowed for

Figure 2. Amount of alkaline phosphatase (AP) bound on a PEGcoated polystyrene (PS) surface or an uncoated polystyrene surface, as a function of the benzophenone (Bz) and AP concentrations in solution and (A) in the presence or (B) in the absence of UV illumination during coupling. The amount of bound AP is reported as the enzymatic activity observed for an equivalent amount of dissolved AP. Error bars show the standard deviation (n = 3).

conjugation of the capture antibody while maintaining the low protein binding properties of the underlying PEG coating (Figure 3, “On PEG-Coated PS”). Immobilization of active capture antibody was verified by a strong increase in the amount of adsorbed detection antibody-HRP when antigen was added in the ELISA protocol compared to the control without added antigen. The photoimmobilization reaction was strongly dependent on the UV illumination time with longer exposure times yielding higher antigen specific signals with some

Figure 3. ELISA assays prepared and performed on PEG-coated or uncoated PS surfaces. The influence of UV illumination time and Bz concentration on the coupling of 20 μg/mL capture antibody was explored for Bz concentrations of (A) 0, (B) 12.5, and (C) 62.5 μg/ mL. The resulting assay response was probed in the presence or absence of added antigen using an HRP-conjugated detection antibody (IgG-HRP). 896

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surfaces were illuminated by UV light in the presence of dissolved antibodies but absence of dissolved Bz, supporting the presence of active immobilized Bz in the underlying PEG coating. Moreover it was also shown that the PEG coating is effective at limiting nonspecific adsorption of all three antibodies used (capture, antigen, and detection antibodies; ESI, Figure S1). Antibody immobilization was also verified using X-ray photoelectron spectroscopy (XPS) that provides information on the surface chemistry. The polystyrene substrate is purely hydrocarbon, while surfaces coated with Bz and PEG showed the presence of both oxygen and nitrogen as expected from their elemental composition (Figure 5). The XPS analysis with

concomitant increase in nonspecific signals. A Bz concentration of 12.5 μg/mL exposed for 30 min yielded the best balance between high antigen sensitivity and low nonspecific response (ESI, Table S1). Moreover, the capture antibody concentration was shown to be optimal at 20 μg/mL or above to achieve high specific signal intensity. The capture antigen could be conjugated onto the surface even without Bz addition, which can be explained by photoactive Bz being immobilized in the PEG layer from the first coating with PEG and Bz (Figure 1). Omission of the PEG coating before capture antibody immobilization (Figure 3, “On PS/0 min UV”) resulted in high signals both with and without the inclusion of antigen in ELISA protocol, most likely due to high nonspecific adsorption of the peroxidase-conjugated detection antibody. The amount of nonspecifically adsorbed detection antibody on the bare PS surfaces in the absence of UV illumination decreased with increasing Bz concentrations, possibly due to the amphiphilic Bz molecule acting as an unintended blocking agent. Functional ELISAs Result from Photoimmobilizing Capture Antibody on Low-Binding PEG. ELISA calibration curves were determined using two different concentrations of capture antibody together with Bz (12.5 μg/mL) on PEGcoated substrate surfaces. An equivalent experiment without exposure of the capture antibody and Bz to UV light was performed as a control. The UV illuminated samples showed an apparently linear (lower part of a sigmoidal) dependence on the logarithm of the antigen concentration as expected for an ELISA type assay (Figure 4A). The limit of detection (LOD),

Figure 5. XPS analysis of the elemental composition on sample surfaces prepared with or without a PEG coating (“PEG”), 100 μg/mL capture antibody (“Capture IgG”), 12.5 μg/mL Bz (“Bz”), and 30 min UV illumination (“UV”). Carbon (not included in the graph) constitutes the remaining part to 100 atom % for each sample.

a probing depth of 5−10 nm resulted in a measured oxygen content of approximately 10 atom % on the PEG coated surface before further treatment, indicative of a PEG layer thickness substantially smaller than the probing depth. In a former publication, we investigated PEG-silane coatings on silicon surfaces by XPS and X-ray reflectometry to establish a correlation between the measured atomic composition and the layer thickness given a homogeneous surface coating and a known photoelectron escape length through the PEG layer.41 Applying the equivalent formalism to the measured oxygen concentration in the present report yields an estimated film thickness of 14 Å for a 750 Da PEG compared to 16 Å in our former work on silicon using 250−400 Da PEG-silanes. The apparently lower PEG layer density using photoimmobilization is still sufficient to strongly reduce protein adsorption as demonstrated in detail in our previous work.32 Thus, the photoimmobilization process is unlikely to result in significant radical induced degradation of the PEG coating. Conjugation of the antibody onto the PEG-coated surface with Bz (12.5 μg/ mL) and UV illumination (30 min) resulted in the appearance of a sulfur signal from the cysteine moieties of the proteins. Moreover, the nitrogen amount also increased compared to samples without Bz, most likely both from nitrogen in the protein backbone, its side groups, and from the Bz itself. Samples without an underlying PEG coating showed a very large increase in both sulfur and nitrogen with or without UV illumination or the addition of Bz, which indicates that the detected signals are primarily from nonspecifically adsorbed

Figure 4. ELISA standard curves resulting from immobilization of capture antibody on a PEG-coated PS surface. The influence of capture antibody concentration was investigated for surfaces where the capture antibody was immobilized with Bz (12.5 μg/mL) and (A) 30 min UV illumination or (B) without UV illumination. Each data point shows the average result and its standard deviation for three samples.

defined as the background signal +3× the standard deviation of the control, was 13 and 42 ng/mL for 100 and 20 μg/mL capture antibody, respectively. Samples not exposed to UV light during capture antibody immobilization resulted in very small detected signals (Figure 4B). This strongly supports that photochemical processes rather than passive adsorption are responsible for the majority of the capture antibody immobilization. A signal could also be detected when the 897

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protein. We additionally investigated the temporal stability of the underlying photoimmobilized PEG coating since excessive radical exposure may result in PEG degradation. A functional assay probing the coating’s ability to reduce passive adsorption of IgG showed no increase, and possibly a decrease, in the amount of adsorbed protein for the maximum investigated storage time of 37 days (ESI, Figure S2). The ELISA protocol was employed for the detection of the chemotactic cytokine (chemokine) CCL19 as an example of a small protein antigen. Capture antibody for the CCL19 antigen was photoimmobilized at different concentrations on PEGcoated polystyrene using Bz (6.25 μg/mL) and the resulting surfaces were used for detection of CCL19 (Figure 6). The LOD was 0.25 ng/mL antigen for 15 μg/mL capture antibody. Interference was only observed at very high CCL19 concentrations (100 ng/mL). Figure 7. Proteins (BSA-fluorescein) micropatterned onto PEGcoated polystyrene in the shape of our university logo (“DTU”) in two different sizes. BSA-fluorescein was spatially selectively conjugated to the surface using Bz as photosensitizer and patterned light exposure in a projection lithography system. The Technical University of Denmark grants the right to use the DTU logo as an example in this article. No other use is permitted.

lithography system, either for patterning of single proteins or for multiple protein types on different areas, simply by employing different coating solutions and masks. This may be highly useful in both lab-on-a-chip systems and for specific cell capture applications.

Figure 6. ELISA standard curves for the cytokine CCL19 using different capture antibody concentrations photoimmobilized with Bz on PEG-coated PS. Each data point shows the average result and its standard deviation for three samples.

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ASSOCIATED CONTENT

S Supporting Information *

Protein Micropatterns can be Photoimmobilized on Low-Binding PEG Coatings. BSA-fluorescein was used to demonstrate that proteins can be micropatterned on a PEGcoated polymer surface using patterned light exposure. BSA is known to exhibit high nonspecific adsorption to many polymer surfaces, thus, calling for an underlying PEG coating of high quality to minimize unwanted adsorption outside the illuminated areas. The coupling of BSA-fluorescein (100 μg/ mL) together with Bz (100 μg/mL) was performed by illumination for 20 min in a home-built projection lithography system using a chrome-on-glass shadow mask with our university logo. Fluorescence confocal microscopy of the resulting protein patterns revealed well-defined microscopic patterns of proteins reflecting the mask motif with micrometer scale resolution (Figure 7). The smallest measured line width is 4 μm.

Quantitative results from optimization of the protein coupling parameter and influence of the photoimmobilized PEG coating on passive protein adsorption. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSION Different protein types can be covalently bound to a low protein binding coating on polymer surfaces with an easy one step photochemical reaction in aqueous buffer. The conjugation of the three types of proteins was verified with enzymatic and ELISA assays as well as XPS and fluorescence microscopy. Both enzymes and antibodies retained their biological activity after conjugation. Moreover, we showed two examples of ELISA with a fast capture antibody conjugation and without a protein blocking step. This improvement could significantly lower the time required to make an ELISA assay. The photoimmobilization process also facilitates patterning of proteins onto specific areas of the low adsorption surface by use of a projection

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +45 4588 7762. Tel.: +45 4525 8161. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Anne Henriksen for technical assistance in constructing the projection lithography setup. We acknowledge financial support from the Danish Advanced Technology Foundation through the PILOC Project (Grant 064-2010-1) and the CELLGIGS Project (Grant 25-2011-5).

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NOTE ADDED AFTER ASAP PUBLICATION This article posted ASAP on February 19, 2014. The paper was originally published with two versions of the same Supporting Information; the older version has been deleted. The correct version posted on February 24, 2014.

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